CM10 Arrays Research Articles

Danger-recognizing proteins, β-defensin-128 and histatin-3, as potential biomarkers of recurrent coronary events.

Conventional risk factors have limited ability to predict recurrent events in subjects with first-time coronary artery disease(CAD). This aim of this study was to identify novel biomarkers using comparative global proteome analysis to improve the risk assessment for recurrent coronary events. We used samples from phase-I of the Indian Atherosclerosis Research Study(IARS), consisting of 2,332subjects, of whom 772were CAD-affected subjects, including 152with recurrent events identified during a 5-year follow-up period. Global proteome analysis was performed on serum samples of 85subjects with recurrent coronary events and 85age- and gender-matched subjects with first-time CAD using surface-enhanced laser desorption ionization time-of-flight mass spectrometry with CM10 arrays. TagIdent was used for protein identification followed by validation by western blot analysis and ELISA. Data were analyzed by logistic analysis, Cox-regression, hazards ratio, C-statistics and combined-marker risk score using SPSS version-17 and R-package version-2.13.0 software. We identified 16significantly differentially expressed protein peaks. Of these, 2peaks corresponding to m/z8588 and1864 were identified as β-defensin-128 and histatin-3, belonging to the danger-recognizing peptide family, which exhibited a significant independent association with recurrent events(odds ratios of7.49 and1.4, respectively). C-statistics improved significantly from 0.677 for conventional risk factors alone to 0.800(p-value=0.001) in combination with β-defensin-128 and histatin-3 with a hazards ratio of1.833. A combined risk score of β-defensin-128 and histatin-3 could reclassify 112out of the 170subjects into intermediate- and high-risk groups. On the whole, our data indicate that β-defensin-128 and histatin-3 may be potential biomarkers whch may be used to improve risk the stratification for recurrent coronary events.

0132 : Usefulness of multiple proteinchip arrays for proteomic profiling using surface enhanced laser desorption ionization – time of flight – mass spectrometry (SELDI-TOF-MS)

Multiple proteinchip arrays have been developed to selectively bind proteins with diverse physical and chemical properties before the profiling step with SELDI-TOF-MS. However, the additional value of each array is poorly described. A proteomic analysis has been performed in plasma of 198 patients with chronic heart failure with a left ventricular ejection fraction <45%. Plasma samples were profiled with CM10 (Weak Cation Exchanger) and H50 (Hydrophobic) proteinchip arrays in a PBS 4000 SELDI-TOF-MS (BioRad Laboratories). To ensure sufficient coverage of the entire mass range, the acquisition settings for low-mass (LM, 2500-30000 Da) and high-mass (HM, >20000 Da) m/z peaks were optimized separately. Correlation between peaks was analysed face to face by the test of Pearson. We detected 203m/z peaks: 109 (52 LM and 42 HM) peaks with the CM10 array and 94 (69 LM and 40 HM) peaks with the H50 array. Among the peaks detected on the H50 array, 28 LM and 8 HM peaks were also present on the CM10 array. In the mass range 20000-30000 Da, peaks can be detected with both, LM and HM acquisition settings. We found 6 of the 13 HM peaks on the CM10 array and 7 of the 15 HM peaks on the H50 array also detected with LM settings. 093We then analysed the correlation among the 203m/z peaks detected with both types of arrays. We found that 56 (27.5%) peaks were highly correlated with at least one other peak with a correlation coefficient r>0.9. Altogether, 30 out of these 56 peaks were correlated with 1 other peak, 14 with 2 other peaks, 7 with 3, 3 with 4, 1 with 5 and 1 with 6 other peaks. These highly correlated peaks may correspond to a unique protein. Profiling with multiple proteinchip arrays provides data with high redundancy and colinearity. This finding may be useful for chosen the SELDITOF- MS peaks to be purified and identified. In addition, a unique array (CM10) may be sufficient to obtain a relevant profiling of plasma proteins.

Proteomic profiling of heterotopic heart-transplanted rats using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry: Potential biomarkers and drug targets

Methotrexate and rapamycin demonstrate an additive effect in prolonging cardiac allograft survival in a major histocompatibility complex mismatched rat model. The present study aimed to identify functional proteins involved in the allograft-protective effects of these two agents and reveal potential diagnostic markers for treating rejection. Serum samples from heterotopic heart-transplanted LEW(RT-1(1)) rats (either without immunosuppressive treatment or treated with methotrexate alone, rapamycin alone, or methotrexate and rapamycin combined) were analysed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Protein profiles obtained using a weak cation exchange ProteinChip® CM-10 array were then analysed using ProteinChip® Software. Of 28 rejection-related proteins identified, isoelectric point and mass information from two potential candidate proteins matched information from the UniProtKB/Swiss-prot database, suggesting them to be complement component C3f fragment and complement component 4A (C4A, anaphylatoxin). Proteomic analysis revealed 28 proteins as potential diagnostic markers of tissue rejection. Of these, 11 proteins may represent targets relating to the additive effects of methotrexate and rapamycin. Two protein peaks, with mass-to-charge ratios of 1950 Da and 8577 Da, may have potential for use in post-transplant diagnosis of rejection.

Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry: A tool to predict pork quality

Expression of water soluble proteins of fresh pork Longissimus thoracis from 4 pure breed pigs (Duroc, Large White, Landrace, and Piétrain) was studied to identify candidate protein markers for meat quality. Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) was used to obtain the soluble protein profiles of Longissimus thoracis muscles. The pure breeds showed differences among the studied meat quality traits (pHu, drip loss, androstenone, marbling, intramuscular fat, texture, and moisture), but no significant differences were detected in sensory analysis. Associations between protein peaks obtained with SELDI-TOF-MS and meat quality traits, mainly water holding capacity, texture and skatole were observed. Of these peaks, a total of 10 peaks from CM10 array and 6 peaks from Q10 array were candidate soluble protein markers for pork loin quality. The developed models explained a limited proportion of the variability, however they point out interesting relationships between protein expression and meat quality.

Identification of serum proteins involved in pancreatic cancer cachexia

AimsTreatment of cachexia requires pharmacological intervention which, in turn, requires knowledge of the mediators and processes. Cachexia markers that are specifically expressed in pancreatic cancer and secreted into the blood circulation have yet to be identified. The aim of our study was to investigate the serum protein profiles and protein alterations associated with cachexia and to identify potential disease protein biomarkers indicative for this syndrome. Main methodsSerum samples from cachectic and non-cachectic patients undergoing pancreatic cancer (PaCa) surgery and controls were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). The identity of detected discriminatory markers was determined by a combination of protein fractionation, chromatographic purification steps, gel electrophoresis, and mass spectrometry. Key findingsUsing Cu-IMAC array and CM-10 array based SELDI-TOF-MS. we identified eleven up- and four down-regulated proteins associated with cachexia. CiphergenExpress analysis revealed four disease-associated protein features (38559Da, 9138Da, 8925Da and 3358Da) that were elevated by a factor of 2.3, 1.7, 1.4 and 1.4, respectively. Zinc-α2-glycoprotein (ZAG), apolipoproteins apo C-II and apo C-III and glucagon-like peptide-1 (GLP-1) were identified as markers for PaCa-associated cachexia syndrome. ZAG levels were additionally evaluated in serum and tissue samples by ELISA and immunohistochemistry and the obtained data confirmed the SELDI-TOF-MS results. SignificanceThe identified proteins could be routinely and reliably measured in the serum of patients and provide an elegant non-invasive approach for early diagnosis of cachectic pancreatic cancer patients. Controlling ZAG and GLP-1 activity could be beneficial in the management of cancers and cachexia-induced conditions.

Sample handling for mass spectrometric proteomic investigations of human urine

Because of its non-invasive sample collection method, human urine is an attractive biological material both for discovering biomarkers and for use in future screening trials for different diseases. Before urine can be used for these applications, standardized protocols for sample handling that optimize protein stability are required. In this explorative study, we examine the influence of different urine collection methods, storage temperatures, storage times, and repetitive freeze-thaw procedures on the protein profiles obtained by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Prospectively collected urine samples from 11 women were collected as either morning or midday specimens. The effects of storage temperature, time to freezing, and freeze-thaw cycles were assessed by calculating the number, intensity, and reproducibility of peaks visualized by SELDI-TOF-MS. On the CM10 array, 122 peaks were detected and 28 peaks were found to be significantly different between urine types, storage temperature and time to freezing. On the IMAC-Cu array, 65 peaks were detected and 1 peak was found to be significantly different according to time to freezing. No significant differences were demonstrated for freeze-thaw cycles. Optimal handling and storage conditions are necessary in clinical urine proteomic investigations. Collection of urine with a single and consistently performed protocol is needed to reduce analytical bias. Collecting only one urine type, which is stored for a limited period at 4°C until freezing at -80°C prior to analysis will provide the most stable profiles.

Searching for Potential Biomarkers that can be Used as Biomarkers for Diagnosing Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic systemic inflammatory rheumatic disorder that primarily affects the axial skeleton. To date there is no satisfactory biomarker for diagnosis or prognosis of AS. This study is focused on detection of potential biomarkers for the diagnosis of AS using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) with the ultimate goal of developing a progression biomarker for AS. Protein profiles of sera of 38 AS patients and 38 healthy controls were obtained using ProteinChip arrays. Different arrays and conditions were compared with find out which condition resulted in the optimal discrimination of both groups. Optimal discrimination was reached using weak cation exchange (CM10) and immobilized metal affinity capture coupled with copper (IMAC-Cu(2+)) arrays. Using a multimarker approach on protein profiles obtained with CM10 arrays resulted in a sensitivity of 66% and a specificity of 74%. Using a multimarker approach on protein profiles obtained with IMAC-Cu(2+) arrays resulted in a sensitivity and specificity of 70%. This is the first study that shows that protein profiling in serum using SELDI-TOF-MS can be used as a diagnostic tool for AS. The biomarkers will be validated to confirm if they can be used as progression biomarkers.

New multi protein patterns differentiate liver fibrosis stages and hepatocellular carcinoma in chronic hepatitis C serum samples

To identify a multi serum protein pattern as well as single protein markers using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) for detection and differentiation of liver fibrosis (F1-F2), liver cirrhosis (F4) and hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus (HCV). Serum samples of 39 patients with F1/F2 fibrosis, 44 patients with F4 fibrosis, 34 patients with HCC were applied to CM10 arrays and analyzed using the SELDI-TOF ProteinChip System (PBS-IIc; Ciphergen Biosystems) after anion-exchange fractionation. All patients had chronic hepatitis C and histologically confirmed fibrosis stage/HCC. Data were analyzed for protein patterns by multivariate statistical techniques and artificial neural networks. A 4 peptide/protein multimarker panel (7486, 12,843, 44,293 and 53,598 Da) correctly identified HCCs with a sensitivity of 100% and specificity of 85% in a two way-comparison of HCV-cirrhosis versus HCV-HCC training samples (AUROC 0.943). Sensitivity and specificity for identification of HCC were 68% and 80% for random test samples. Cirrhotic patients could be discriminated against patients with F1 or F2 fibrosis using a 5 peptide/protein multimarker pattern (2873, 6646, 7775, 10,525 and 67,867 Da) with a specificity of 100% and a sensitivity of 85% in training samples (AUROC 0.976) and a sensitivity and specificity of 80% and 67% for random test samples. Combination of the biomarker classifiers with APRI score and alfa-fetopotein (AFP) improved the diagnostic performance. The 6646 Da marker protein for liver fibrosis was identified as apolipoprotein C-I. SELDI-TOF-MS technology combined with protein pattern analysis seems a valuable approach for the identification of liver cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C. Most probably a combination of different serum markers will help to identify liver cirrhosis and early-stage hepatocellular carcinomas in the future.

Discovery of new rheumatoid arthritis biomarkers using the surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry ProteinChip approach

To identify serum protein biomarkers specific for rheumatoid arthritis (RA), using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. A total of 103 serum samples from patients and healthy controls were analyzed. Thirty-four of the patients had a diagnosis of RA, based on the American College of Rheumatology criteria. The inflammation control group comprised 20 patients with psoriatic arthritis (PsA), 9 with asthma, and 10 with Crohn's disease. The noninflammation control group comprised 14 patients with knee osteoarthritis and 16 healthy control subjects. Serum protein profiles were obtained by SELDI-TOF-MS and compared in order to identify new biomarkers specific for RA. Data were analyzed by a machine learning algorithm called decision tree boosting, according to different preprocessing steps. The most discriminative mass/charge (m/z) values serving as potential biomarkers for RA were identified on arrays for both patients with RA versus controls and patients with RA versus patients with PsA. From among several candidates, the following peaks were highlighted: m/z values of 2,924 (RA versus controls on H4 arrays), 10,832 and 11,632 (RA versus controls on CM10 arrays), 4,824 (RA versus PsA on H4 arrays), and 4,666 (RA versus PsA on CM10 arrays). Positive results of proteomic analysis were associated with positive results of the anti-cyclic citrullinated peptide test. Our observations suggested that the 10,832 peak could represent myeloid-related protein 8. SELDI-TOF-MS technology allows rapid analysis of many serum samples, and use of decision tree boosting analysis as the main statistical method allowed us to propose a pattern of protein peaks specific for RA.